The Ras Superfamily of GTPases
Proteins regulating Ras and its relatives have been reviewed in Boguski et al. (Nature 366:643-654 (1993)), summarized below. Ras proteins and their relatives are key in the control of normal and transformed cell growth. Small GTPases related to Ras control a wide variety of cellular processes which include aspects of growth and differentiation, control of the cytoskeleton and regulation of cellular traffic between membrane bound compartments. These proteins cycle between active and inactive states bound to GTP and GDP. This cycling is influenced by three classes of proteins that switch the GTPase on, switch it off, and prevent it from switching. Further, the intracellular location of the GTPase can be controlled by another class of regulatory protein. The GTP-bound form of the GTPase is converted to the GDP-bound form by an intrinsic capacity to hydrolyze GTP. This process is accelerated by a GTPase-activating protein (GAP). Activation involves the replacement of GDP with GTP. This event is mediated by proteins designated guanine nucleotide exchange factors (GEF) or guanine nucleotide releasing protein (GNRP) and guanine nucleotide dissociation stimulator (GDS). The process is inhibited by guanine nucleotide dissociation inhibitors (GDI). Further, membrane anchoring of the GTPase is critical for proper function and is regulated, among other enzymes, by prenyltransferases.
The Ras superfamily of GTPases can be roughly divided into three main families. The first family is the “true” Ras protein, each of which has the ability to function as an oncogene following mutational activation. These proteins transmit signals from tyrosine kinases at the plasma membrane to a cascade of serine/threonine kinases, which deliver signals to the cell nucleus. Constitutive activation of the pathway contributes to malignant transformation. The second group is the Rho/Rac protein subgroup, involved in organizing the cytoskeleton. Rac is required for membrane ruffling induced by growth factors and the formation of actin stress fibers requires Rho. In yeast, the CDC42 product controls cell polarity, another process in which actin is involved. In addition, Rac proteins are components of the NADPH oxidase system that generates superoxide in phagocytes. A third family is the Rab protein family. Members of this group regulate membrane trafficking, i.e., transport of vesicles between different intracellular compartments.
In addition to the three major families, further subgroups exist, exemplified by Ran and Arf. Ran proteins are nuclear GTPases involved in mitosis. Arf (ADP-ribosylation factor) proteins are necessary for ADP-ribosylation of Gsa (the GTPase subunit of s-type heterotrimeric G-proteins) by cholera toxin and are thought to be involved in membrane vesicle fusion and transport.
Ras GEFs are proteins that activate Ras proteins by exchanging bound GDP for free GTP. These include Ras GRF, MmSosI, DnSoS, Ste6, Cdc25, Scd25, Lte1, and BUD5. The loss of GEF function can be complemented by mutations that constitutively activate the Ras proteins or, in some cases, by a loss of GAP activity. GEFs first associate with the GDP-bound form of the GTPase. GDP dissociates from this complex at an increased rate leaving the GEF bound to the empty GTPase. GTP then binds immediately, effecting GEF dissociation and leaving the GTPase in active form. Accordingly, a stable complex can exist between GEF and GTPase in the absence of nucleotide. Thus, GEFs recognize both GDP and GTP-bound forms of Ras in vitro and in vivo.
Dominant negative Ras mutants exist that block normal Ras activation. These have reduced affinity for GTP and may be defective in the final step of the exchange process, i.e. displacement of GEF by GTP. Accordingly, these mutants sequester GEF into a dead-end complex and are useful to remove GEF activity from cells so that activation of endogenous Ras proteins cannot occur. However, Ras may also be activated by inhibiting GAP activity without the need for GEF.
GEFs also include rap GEF. It is 20-fold more active on Ral A and Ral B than on members of the Ras, Rho/Rac and Rab GTPase families.
GEFs also include rap GEF. Cell polarity and budding in yeast involve GTPases of the Rap and Rho subgroup. A GEF specific for mammalian Rap proteins remains to be identified. Rap has the ability to interfere with Ras signaling by blocking activation of RAF and the serine/threonine kinase cascade.
GEFs also include Rho/Rac GEFs. GEFs specific for Rac and Rho proteins include, but are not limited to, Cdc24, Dbl, Vav, Bcr, Ras GRF, and ect 2. The human Dbl has been shown to act as a GEF for CDC42Hs (the human homolog of CDC42 is known as G25K) and on Rho. Further, Dbl binds several Rac/Rho-like proteins in vitro.
smg GDS (small GTP-binding protein) was originally described as a GEF for mammalian Rap proteins. It also promotes nucleotide exchange on Rho and Rac proteins. The protein works efficiently only on isoprenylated proteins. Ras and Rho/Rac proteins are modified by different isoprenoid moieties. Rho/Rac proteins receive 20-carbon geranylgeranyl groups.
Guanine nucleotide dissociation inhibitors (GDIs) include rab GDI. The protein affects the rate of GDP dissociation from Rab proteins. It inhibits GDP/GTP exchange and prevents the GDP-bound form from binding to membranes. These activities depend on the C-terminal geranylgeranyl group, at least of Rab3A.
Rho GDI was first identified as a factor capable of inhibiting dissociation of GDP from post-translationally modified Rho proteins. It has the ability to remove Rho proteins from cellular membranes in cell-free systems. This indicates that it could regulate the available Rho proteins associated with membranes or facilitate movement of Rho from one membrane compartment to another. Rac proteins bound to Rho GDI have also been identified as components of the NADPH oxidase system that generates oxygen radicals in activated phagocytes. Rac and Rho GDI form a heterodimer required for oxidase stimulation in vitro. Along with two other cytosolic factors, the components assemble into a membrane-bound complex which uses electrons from NADPH to generate superoxide anions. Recombinant Rac proteins in their GDP-bound state can replace the requirement for Rac and Rho GDI in this system. This indicates that Rho GDI can recognize the GTP-bound form of Rac and protect it from Rac GAPs.
GTPase-activating proteins are disclosed in Table 1 in Boguski et al., above. These include Ras GAP proteins. These proteins have low intrinsic GTPase activity and their inactivation is dependent on GAP in vivo. Of the Ras GAPs, neurofibromin, p120 GAP, Ira1, and Ira2 also have specificity for Rac. Of the rap GAP family, Rap1GAP also has specificity for Rac. Rho/Rac GAPs with specificity for Rac include Bcr, N-chimerin, rotund, p190, GRB-1/p85a, and 3BP-1.
Ras-like GTPases are targeted to membranes where they act by the post-translational attachment of isoprenoid lipids (or prenyl groups). Prenylation involves the covalent thioether linkage of farnesyl (15-carbon) or geranylgeranyl (20-carbon) groups to cysteine residues near the C-terminus. These reactions are catalyzed by prenyltransferases that differ in their isoprenoid substrates and protein targets. Type 1 geranylgeranyl transferase recognizes a CAAX motif but prefers a leucine residue in the X-position. Substrates include members of Rho/Rac families.
p21-activated protein kinases (PAKs) are activated through direct interaction with the GTPases Rac and Cdc42Hs. These GTPases are implicated in the control of mitogen-activated protein kinase (MAP) kinase c-Jun N-terminal kinase (JNK) and the reorganization of the actin cytoskeleton. Recently, Aronheim et al. (Current Biology 8:1125-1128 (1998)) reported on the biological role of PAK2 and identified its molecular targets. A two-hybrid system, “the Ras recruitment system” was used to detect protein-protein interactions at the inner surface of the plasma membranes. The PAK2 regulatory domain was fused at the carboxy terminus of a Ras mutant protein and screened against a cDNA library. Four clones were identified that interacted specifically with PAK regulatory region and were shown to encode a homolog of the GTPase Cdc42Hs. This protein, designated Chp, showed an overall sequence identity to Cdc42Hs of approximately 52%. Results from microinjection of this protein into cells implicated it in the induction of lamellipodia and showed that it activates the JNK MAP kinase cascade.
Proteins regulating Ras and its relatives have been reviewed in Boguski et al., Nature 366: 643-654 (1993), summarized below. As indicated above, GTPases cycle between inactive and active states bound to GDP and GTP respectively. As indicated above, cycling can be influenced by three different classes of proteins that switch the GTPase on, switch it off, and protect it from switching. Classes of regulatory proteins of Ras-like GTPases include GEF, GDI, and GAP. GEFs catalyze exchange of GDP for GTP. GAPs catalyze conversion of GTP-bound forms back to their inactive GDP states. GDI proteins for Rab and Rho affect nucleotide dissociation and GAP attack and may also be involved in membrane localization and solubility. The intracellular location of the GTPase can be controlled by a fourth class of regulatory protein affecting the regulators with which the GTPase can interact.
Table 1 of Boguski et al. lists various GAPs, the organisms from which they are derived, substrate specificity, and other characterization. These include (in the Table) the following GAPs: RasGAP; Neurofibromin (NF1) with a positive specificity for H-ras, N-ras, K-ras, RAS1 and RAS2 and a negative specificity for Rho, Rac, and Rab; p120GAP with a positive specificity for H-ras, N-ras, K-ras, R-ras, RAS1 and RAS2 and a negative specificity for Rho, Rac and Rab; Gap1 with a positive specificity for Ras1; Ira1 with a positive specificity for RAS and RAS2 and a negative specificity for Rho, Rac and Rab and potentially H-ras; Ira2 with a positive specificity for RAS and RAS2 and a negative specificity Rho, Rac and Rab and potentially H-ras; Sar1/gap1 with a positive specificity for Ras1, RAS1 and Ras2; Bud2 with a positive specificity for Bud1; RapGAP and Rap1GAP with a positive specificity for Rap1A and Rap2 and a negative specificity for Ras, Rho and Rac; Rho/racGAP and Bar with a positive specificity for Ras and CDC42Hs and a negative specificity for Rho and Ras; n-Chimaerin with a positive specificity for Rac and a negative specificity for Rho, CDC42Hs and Ras; rotund locus and p 190 with a positive specificity for Rac, Rho and CDC42Hs and a negative specificity for Ras, GRB-1/p85a and 3BP-1.
RasGAP is one class of GAP. Ras proteins have a very low intrinsic GTPase activity and their inactivation is dependent on GAPs in vivo. For example, some oncogenic mutants of Ras proteins are resistant to GAP-mediated GTPase stimulation and are constitutively blocked in their active GTP-bound states. Yeast contains two RasGAP proteins, IRA1 and IRA2 which contain domains homologous to the human and other mammalian p120-GAPs. In the absence of IRA gene product, yeast RAS proteins accumulate in their GTP-bound state, becoming hyperactive and leading to overproduction of cAMP. In yeast, therefore, RasGAPs are not effectors but serve as negative regulators. NF1 is a human protein defective in von Recklinghausen neurofibromatosis. This protein contains a domain homologous to the catalytic domains of p120-GAP IRA1 and IRA2. It may, in fact, be the mammalian homolog of IRA1 and IRA2. Mutant NF1 alleles are associated with sporadic cancers unrelated to neurofibromatosis or to neural crest tissues. Drosophila contains a protein, 70% identical to neurofibromin. It also contains a distinct RasGAP (referred to as GAP1) that is a component of the Sos tyrosine kinase/Ras1 signalling pathway. Loss of GAP1 stimulates Ras1 function, indicating that it is a negative regulator.
RapGAP is another GAP class. Rap1A is around 50% identical to Ras and, like Ras, binds to p120-GAP and to raf1 by its effector binding domain. Rap1A binds p120-GAP but its GTPase activity is not enhanced by this interaction. Another protein, rap1GAP, is responsible for the Rap1A GTPase activation. Rap1GAP is unrelated to rap1GAP but contains several sites for phosphorylation by Cdc2 and cAMP-dependent kinases. Ras proteins, and most GTPases, depend on a glutamine residue at position 61 (or equivalent) for intrinsic or GAP-mediated GTP hydrolysis. Rap1, however, has a threonine at this position.
Rho/Rac GAP is another class of GAP. A mammalian GAP specific for Rho has been purified and shown to contain a region related to the C-terminal domain of Bcr and to a human brain protein, n-chimaerin. Bcr is a putative RhoGEF. Bcr and n-chimaerin stimulate GTP hydrolysis by the Rho-like proteins Rac1 and Rac2, but not by Rho proteins themselves. This activity is mediated by the C-terminal 401 amino acids of Bcr. This domain does not resemble RasGAP or Rap1GAP. Chimaerin also contains an N-terminal DAG binding motif. Further, a multidomain protein, p90, that binds to p120-GAP and regulates its activity contains a central domain related to a putative DNA binding transcriptional repressor. At the C-terminus, there is a 145 residue region that is related to RhoGAPs.
GTPase activators (GAPs) are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown GAPs. The present invention advances the state of the art by providing previously unidentified human GAPs.